Noonan syndrome (NS) is an autosomal dominant disorder that occurs with a frequency of ~ 1:2,000 live births. Approximately 50% of NS patients contain a gain-of-function mutation in the human PTPN11 gene which encodes for the SH2 domain-containing protein tyrosine phosphatase, SHP-2. NS patients exhibit a diverse array of clinical manifestations, most notably, congenital heart disease (CHD). CHD occurs in up to 80% of NS patients, making PTPN11/SHP-2 mutations the most common non-chromosomal cause of CHD. Therefore, altered tyrosyl phosphorylation underlies the basis for CHD. The broad goal of this research is to uncover the molecular basis for how NS-associated SHP-2 mutations give rise to CHD. Although much work has established that enhanced activation of the Ras/extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway is causal to NS-mediated CHD the precise mechanisms through which NS-associated SHP-2 mutations engage in pathophysiological signaling to Ras/ERK1/2 remains unknown. We propose to identify the direct upstream and downstream targets of NS-associated SHP-2 mutants and determine if these targets are involved in the development of NS-mediated CHD. In the first aim, we have identified that NS-associated SHP- 2 mutants interact preferentially with ITIM-containing transmembrane glycoproteins. We hypothesize that dysregulated membrane proximity by NS-associated SHP-2 mutants engages promiscuous dephosphorylation of substrates that evoke Ras/ERK1/2 signaling. The contribution of these ITIM/NS-SHP-2 interactions to signal to Ras/ERK1/2 will be defined. The substrates involved in NS-mediated Ras/ERK1/2 activity will be identified and characterized for their involvement in NS-associated SHP-2 mutant signaling. In specific aim two, the ITIM containing transmembrane glycoproteins have been identified to be hypertyrosyl phosphorylated in a mouse model of NS. We will identify the NS-induced tyrosine kinase(s) and using a combination of genetic and biochemical approaches determine whether this tyrosine kinase(s) propagates enhanced ERK1/2 activation and subsequently NS-related cardiac defects. The third aim will test the pathophysiological contribution of altered membrane recruitment of NS-associated SHP-2 mutants as a determinant of NS-mediated cardiac defects. We will accomplish this by employing genetic approaches to interfere with the recruitment of NS- associated SHP-2 to the membrane. The completion of these studies will yield new insight into the direct targets of NS-associated SHP-2 mutants in CHD, and may reveal unanticipated roles, for new and established, signaling molecules in this disease. The identification of targets involved in CHD will also reveal new modes of therapeutic strategies in which to treat, and prognostic tools in which to evaluate, NS-related CHD. PUBLIC HEALTH RELEVANCE: Noonan syndrome (NS) is an autosomal dominant developmental disorder that occurs with a frequency of ~1:2,000 live births in the United States. The cause of NS has been mapped to the PTPN11 gene, which encodes for the SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2). This application seeks to understand the signaling mechanisms through which mutated PTPN11 causes NS-related defects in the heart.